高效产孢<em>酿酒酵母</em>在96多孔形式

Summary

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Abstract

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Introduction

在芽殖酵母孢子形成进行了研究减数分裂^1，基因重组²的机制，开发由细胞信号³的控制下，开发⁴的营养控制，转录期间提供见解生物学的许多方面，包括染色体分离的控制发展⁵调控，以及孢子形成⁶考试。孢子形成包括涉及的新的膜隔室的母细胞，随后用保护孢子壁⁶的沉积中形成的新的细胞分裂事件。这些研究，考察形成孢子的细胞经常采取迅速孢子酵母菌株SK1，其可以经受孢子形成的过程中约24小时以相对有效的方式^7,8的优点。虽然对芽殖酵母产孢条件的优化已经被描述^9-13，这些实验S于固体培养基或在孢子形成是利用培养管或瓶中大规模液体培养观察孢子形成。

在这里，我们描述了在96多井板格式形成孢子酵母的方法。我们发现，对于这种方法，曝气是用于同步和有效的孢子形成关键的，并且已经设计技术，以确保有足够的孢子形成的小体积多孔形式。在96多井板格式孢子允许使用高通量技术和多孔板格式，这种筛选使用平铺库^14-16高拷贝抑制优化试剂进行筛选细胞。

Protocol

1.准备产孢注：在本协议中所描述的媒体使用标准配方和方法制成的13,17 表1给出了1升在本协议中所使用的各种媒体的表述。 细菌蛋白胨酵母抽提物</td…

Representative Results

为了评估这个协议中，从在多孔板形成孢子的细胞（如上述）获得的孢子形成的效率进行了比较，在瓶（ 表2）使用较大体积的形成孢子的细胞。使用多孔板的没有达到在瓶中，在〜80％的效率可常规看出形成孢子时见到的高效率。在与适当的曝气（通过玻璃珠或搅拌棒提供的）多孔板形成孢子可以达到足够的孢子形成的效率大于50％，用一个5毫米×2mm的?…

Discussion

在这里，我们提出了一个协议，用于在96多孔形式孢子SK1酵母。曝气是有效孢子形成，这需要使用任一搅拌棒或在每一个玻璃珠孔的关键。当细胞在96多孔板中形成孢子在没有任何一个珠或搅拌棒的振荡培养箱中，细胞不能有效地形成孢子。当细胞没有任何一个珠或在振荡培养箱搅拌棒相比，是在30℃下不搅拌（ 表1形成孢子仅在孢子形成效率的少量增加被看见;在摇对10％17％放置在搅?…

Materials

Nunc 1.3 ml DeepWell Plates	ThermoScientific	260251	Used for sporulation
Nunc 2.0 ml DeepWell plates	ThermoScientific	278743	Used for presporulation growth, step 1.2.3
3 mm glass bead	Fisher	11-312A	Used for sporulation
5 mm x 2 mm stir bar, pack of 12	Fisher	14-511-82	Used for sporulation
96 well frogger	V&P Scientific	VP407	needed for step 1.2
library copier	V&P Scientific	VP381	needed for step 1.2; to be used with the frogger
rectangular petri dish	ThermoScientific	264728	needed for step 1.2
Bacto Peptone	BD	211677	needed for media
Yeast Extract	BD	212750	needed for media
Bacto Agar	BD	212750	needed for media
Dextrose	Fisher	D16-3	needed for media
Potassium Acetate	Fisher	P171-500	needed for media
Glycerol	Fisher	G33-500	needed for media
Black 96 well glass bottom plate	MatTek	PBK96G-1.5.5-F	needed for step 2.4